This invention relates to a method for modulating the activity of the protein p53 in cells by the addition of a peptide or protein having p33ING1 biological activity or a nucleic acid coding for such a peptide or protein.
The following references are cited in the application as numbers in brackets ([ ]) at the relevant portion of the application.
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The disclosure of the above publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if the language of each individual publication, patent and patent application were specifically and individually included herein.
Many cancers originate and progress by accumulating mutations in one or more genes. Such mutations which result in cancer formation can be in proto-oncogenes or in tumor suppressor genes. Mutations in tumor suppressor genes result in loss of function, and therefore act in a recessive fashion to native genes. Oncogenes, in contrast, act in dominant fashion to native alleles and, therefore, are not usually inherited through the germ lines. The tumor suppressor genes, however, are found in inherited predispositions to cancer and are inherited as a dominant predisposition because of the high frequency of a second genetic event such as reduction in homozygosity [1].
Several tumor suppressor genes have been identified. Examples include the Rb gene, which is involved in retinoblastoma and osteosarcoma; p53, which is involved in osteosarcoma and adrenocortical, breast and brain cancers; WT-1, which is involved in Wilms"" tumor, nephroblastoma and neurofibromatosis; adenomatous polyposis coli (APC), which is involved in adenomatous polyposis; and deleted colorectal cancer (DCC), which is involved with a somatic mutation in the colon.
The p53 protein is a transcription factor that enhances the rate of transcription of six or seven known genes that carry out, at least in part, the p53-dependent functions in a cell. These genes include p21, WAF1, Clp1, MDM2, GADD45, Cyclin G, Bax and IGF-BP3. P53 has also been shown to bind to c-Abl and enhance c-Abl""s transcriptional activity. The p53 protein has also been shown to bind to an RNA polymerase II basal transcription factor TFIIH. TFIIH consists of two helicases which are implicated in the disease xeroderma pigmentosum. The Wilms"" tumor suppressor gene product, WT1, has been shown to associate with p53 when both are overexpressed in the same cell.
The human p53 protein contains 393 amino acids and has been divided structurally and functionally into four domains [2]. The first 42 amino acids at the N-terminus constitute a transcriptional activation machinery in positively regulating gene expression. Amino acids 13-23 in the p53 protein are identical in a number of diverse species and certain amino acids in this region have been shown to be required for transcriptional activation by the protein in vivo. The sequence-specific DNA binding domain of p53 is localized between amino acid residues 102 and 292. The native p53 is a tetramer in solution, and amino acid residues 324-355 are required for this oligomerization of the protein. The C-terminal 26 amino acids form an open domain composed of nine basic amino acid residues that bind to DNA and RNA readily with some sequence or structural preferences. There is evidence that the p53 protein requires a structural change to activate it for sequence specific binding to DNA. Deletion of the C-terminus domain activates site-specific DNA binding by the central domain.
Normally, in a cell, the p53 protein is kept at a low concentration by its relatively short half-life. The events or signals that activate p53 are mediated by several stressful events. Several different types of DNA damage can activate p53, including double-stranded breaks in DNA produced by xcex3-irradiation and the presence of DNA repair intermediates after ultraviolet irradiation or chemical damage to DNA. This results in a rapid increase in the level of p53 in the cell and the activation of p53 as a transcription factor. In addition to DNA damage, hypoxia is able to stimulate p53 levels and activate the p53 protein. If ribonucleoside triphosphate pools fall below a critical threshold then p53 is also activated [2].
53 mutations are found in 50-55% of all human cancers. These mutations strongly select for p53 proteins that fail to bind to DNA in a sequence-specific fashion. It is clear that wild-type p53 acts to reduce the incidence of cancers by mediating apoptosis in cells with activated oncogenes. The treatment of neoplasia using radiation and chemotherapy results in extensive DNA damage and the activation of wild-type p53 in those cells. It is becoming clear that p53-dependent apoptosis can modulate the toxic effects of anticancer agents.
It would be advantageous to identify factors or proteins which enhance the activity of the wild-type p53 gene.
The gene ING1 (formerly called p33IG1) described in International Patent Application No. WO97/21809 [3] and in Garkavtsev [12-14], represents a new tumor suppressor gene which is expressed in normal mammary epithelial cells, but only expressed at lower levels in several cancerous mammary epithelial cell lines and is not expressed in many primary brain tumors. The gene produces a 33 kD protein called p33ING1. The amino acid sequence of the p33ING1 related proteins p28ING1 and p26ING1 have been disclosed in U.S. application Ser. No. 09/006,783 filed Jan. 14, 1998.
This invention relates to the discovery that the protein p33ING1 and related proteins, including p28ING1 and p26ING1 bind to p53 in vivo and activate p53 as a transcription factor in acute cotransfection assays. Thus, the invention provides methods for using p33ING1 related proteins to enhance or inhibit the biochemical activity of p53.
The present invention is directed to a method of modulating the activity of p53 in a cell by administering an effective amount of a peptide having p33ING1 biological activity to the cell.
In another method aspect the present invention is directed to a method of modulating the activity of p53 in a cell by administering an effective amount of a nucleic acid encoding a peptide having p33ING1 biological activity to the cell. The nucleic acid may be DNA or RNA.
In a further aspect the present invention is directed to a method for isolating p53 comprising obtaining a biological sample containing p53; contacting said biological sample with a peptide having p33ING1 biological activity under conditions wherein the p53 binds to the peptide; and isolating the p53 bound to the peptide.
In another aspect, this invention is directed to a method for detecting the presence of p53 in a sample comprising obtaining a biological sample suspected of containing p53; contacting said biological sample with a peptide having p33ING1 biological activity under conditions wherein the p53 binds to the peptide; and detecting the presence of p53 bound to the peptide.
In yet a further aspect, the invention provides methods of diagnosing a malignant or premalignant condition in a human by obtaining a cell or tissue sample from the human; assaying the sample to determine ING1 gene expression in the sample; comparing ING1 gene expression in the sample with ING1 gene expression in a nonmalignant human cells or tissue; and assaying the sample to determine p53 gene expression in the sample; wherein a malignant or premalignant condition is diagnosed when both p53 and ING1 gene expression in the sample is less than p53 or ING1 gene expression in the nonmalignant human cells or tissues.
In a still further aspect, the invention provides methods of screening a human sample to detect a malignant or premalignant condition by obtaining a cell or tissue sample from the human; assaying the sample to determine ING1 gene expression in the sample; comparing ING1 gene expression in the sample with ING1 gene expression in a nonmalignant human cells or tissue; and assaying the sample to determine p53 gene expression in the sample; wherein a malignant or premalignant condition is detected when both p53 and ING1 gene expression in the sample is less than p53 or ING1 gene expression in the nonmalignant human cells or tissues.
In still another aspect, the invention provides methods of assessing a human sample to determine a risk of developing a malignant or premalignant condition by obtaining a cell or tissue sample from the human; assaying the sample to determine ING1 gene expression in the sample; comparing ING1 gene expression in the sample with ING1 gene expression in a nonmalignant human cells or tissue; assaying the sample to determine p53 gene expression in the sample; wherein a risk for developing a malignant or premalignant condition is determined to exist when both p53 and ING1 gene expression in the sample is less than p53 or ING1 gene expression in the nonmalignant human cells or tissues.
In a yet still further aspect, the invention provides pharmaceutical compositions useful for modulating p53 activity comprising an effective amount of at least one selected from the group consisting of a peptide or protein having p33ING1 biological activity and a nucleic acid coding for a peptide or protein having p33ING1 biological activity, wherein said effective amount is sufficient to provide effective modulation of p53 activity.